The identification of sperm has been and remains the single most accurate confirmatory test for the determination of sexual activity. Unfortunately, the microscopic examination of slides to identify sperm cells is one of the most tedious and time consuming activities in the forensic biology laboratory.
One of the main reasons for the huge backlog in DNA cases awaiting analysis in forensic laboratories across the country and the world is that the initial step in making the DNA analysis is the manual microscopic identification of the sperm cells found in the smears taken in each rape case. Automated DNA detection is not sensitive enough to give an effective result in situations where less than about 50 sperm heads are present. Low sperm count can be the result of many factors, for example, the victim may not be seen in the hospital immediately after the attack.
Evidence from sexual assaults constitutes the most common DNA sample type tested by crime laboratories. Microscopic identification of spermatozoa is one of two common confirmatory tests for semen and the only confirmatory test for the presence of sperm cells. Quantification through sperm detection also provides an important control for the amount of recovered male DNA.
Extraction and purification of DNA from sexual assault evidence is accomplished via a so-called differential extraction procedure. This produces two separate samples; one enriched in DNA from sperm cells, the other containing DNA from all other cells, for example, cells from the victim's vaginal epithelium. Of course, the sperm cell fraction typically is the more informative of the two, enriched in the DNA of the perpetrator or man from whom the sperm arose.
It is critical for laboratory personnel to be able to estimate the number of sperm cells entering the extraction procedure to be sure the sperm cell DNA has been recovered successfully. That is, low numbers of sperm cells entering the procedure should result in a low yield of DNA in the sperm cell fraction, and vice versa. Omitting the microscopic examination prevents the analyst from making this key comparison.
Microscopic identification of sperm can be an extremely tedious and time-consuming endeavor, however. When few sperm are present, it is not unusual for an analyst to spend an hour or more searching each of multiple individual slides for identifiable sperm cells.
After locating sperm, the analyst can then mark the position of sperm cell heads or sperm cells with tails still attached. The finding of sperm cells with tails can be used in court to collaborate the timeline of an alleged crime. The presence of sperm cells may be used to support DNA findings and as evidence that the match was not just from casual contact. Both may be critical to a rape case when DNA matches are found and to support reports of a crime.
Cases in which there is an allegation of rape but in which no sperm are immediately found are much more time consuming for the technician. A highly skilled technician will spend no more than two hours searching a slide for a single sperm head, often stopping the search far earlier because of the backlog of casework. A time limit of two hours is a common policy however time limits may be more or less in different laboratory settings. In a typical rape case three to ten slides are examined. The manual microscopic examination of slides showing a low sperm count is extremely tedious. In addition, it is a great deal more difficult to identify sperm cells when the slide also includes a high density of debris that is similar in appearance to sperm cells such as white blood cells or dense layers of epithelial cells.
State crime laboratories across the nation typically dedicate multiple microscopes operated by multiple technicians within the lab to the manual search for sperm cells in smears from rape cases. Even private forensic laboratories, which generally have the most advanced automation available, still rely on manual examination of slides to identify sperm cells as the first step in analysis of evidence in a rape case.
Presently, slides used to identify sperm cells are typically stained with what is commonly called a Christmas tree stain. This staining technique stains the head of the sperm cell red and the tail of the sperm cell green. The staining technique is scientifically known as Kernechtrot-Picroindigocarmine staining (KPICS). Research has shown that this stain is currently the most accurate staining technique for detection of sperm cells especially in cases where the smear has been created days after the alleged rape.
Thus, the forensic laboratory would benefit greatly from an automated system which could speed the search for sperm cells on smeared slides and reduce the time that laboratory technicians need to dedicate to the identification of sperm cells.